Landmark analysisof DNMT3A mutations in hematological malignancies

Blood Cancer Journal - Letter to the Editor

Characteristics of DNMT3A mutations in AML

DNMT3A mutations in AML were observed predominantly in the conserved residues including the methyltransferase domain (MTase), zinc finger domain (ZNF), and proline-tryptophan-tryptophan-proline domain (PWWP), and almost all mutations were heterozygous.1 The majority of mutations (range: 80.0% - 91.6%) were located in the MTase domain and, interestingly, more than 50% (range: 54.5 - 87.5%) of aberrations affected the amino acid residue Arg882.1-3

Material

Bone marrow and/or peripheral blood samples were referred to the MLL Munich Leukemia Laboratory for diagnostic assessment between 09/2005 and 06/2012. All samples were obtained from untreated patients.All patients gave their consent to genetic analyses, and agreed to the performance of research studies. The study was approved by the Internal Review Board and adhered to the Declaration of Helsinki.

Methods

Cytomorphology, immunophenotyping, FISH, and chromosome banding analyses followed previous descriptions.4, 5 For molecular analyses, mononuclear cells purified from peripheral blood (n=82) or bone marrow (n=429) by Ficoll density enrichment were used.Transcript-IDs and primer sequences used were published elsewhere.6

NGS data was analyzed using the GS Variant Analyzer Software 2.5.3 (454 Life Sciences, Branford, USA) and Sequence Pilot version 3.5.2 (JSI Medical Systems, Kippenheim, Germany). Known single-nucleotide polymorphisms were filtered out using NCBI dbSNP version 135.7Deleterious effects of mutations on DNA level were predicted by the MutationTaster software.8

Statistical analyses were performed using SPSS version 19.0.0 (IBM Corporation, Armonk, NY). Dichotomous variables were compared between different groups using the Fisher’s exact test and continuous variables by Student’s T-test. Results were considered significant at P<0.05. Overall (OS) and event-free survival (EFS) were analyzed by the Kaplan Meier estimator and compared by log rank test. Differences in survival according to continuous variables were analyzed by Cox regression analysis. The reported p-values are two-sided.

Associations with additional molecular markers

In CMML, theDNMT3A mutations showed no significant associations with other molecular markers (patients analyzed for mutations in ASXL1: n=100, CBL: n=101,EZH2: n=93, JAK2: n=103, KRAS: n=80,NRAS: n=74, RUNX1: n=102, SRSF2: n=102, TET2: n=82). However, we identified 4/7 CMML cases showing at least one additional mutation in the genes investigated. MDS cases were characterized for RUNX1 (n=34), SF3B1 (n=35), JAK2 (n=42), and FLT3-ITD (n=35) mutations without any significant association to DNMT3A mutations. Eight of 15 cases (53.3%) were observed with an additional mutation. In T-ALL, DNMT3A mutations showed no significant association with RUNX1 (n=93), NOTCH1 (n=95), FBXW7 (n=94), PTEN (n=90), and NPM1 (n=90) mutations. However, only one sample had no further mutation within the analyzed genes.

Supplementary Table 1

The table summarizes the clinical characteristics for the four different entities (AML, CMML, MDS, and T-ALL) analyzed in this study.

de novo CN-AML
Patients (n=194)
Gender
male / 107 (52.2%)
female / 98 (47.8%)
Age (years)
median / 65.2
range / 21-100.4
FAB subtype
M0 / 8
M1 / 50
M2 / 65
M4 / 37
M5 / 7
M6 / 9
M7 / 1
missing data / 17
WBC count x 10^9/L
median / 18.3
range / 1-249.5
no data available / 31 (16.0%)
Platelet count x 10^9/L
median / 76
range / 7-540
no data available / 39 (20.1%)
Hemoglobin level (g/L)
median / 9.3
range / 4.9-16.3
no data available / 39 (20.1%)
MDS
Patients (n=115)
Gender
male / 72 (62.6%)
female / 43 (37.4%)
Age (years)
median / 70.7
range / 27.6-89.1
FAB subtype
RA / 10
RARS / 23
RARS-T / 21
RAEB-1 / 19
RAEB-2 / 12
RCMD / 10
RCMD-RS / 10
5q- / 10
Karyotype
normal / 74 (64.3%)
aberrant / 41 (35.7%)
WBC count x 10^9/L
median / 5.7
range / 1-50
no data available / 12 (10.4%)
Platelet count x 10^9/L
median / 245
range / 28-1,068
no data available / 13 (11.3%)
Hemoglobin level (g/L)
median / 10
range / 6.9-15.5
no data available / 13 (11.3%)
CMML
Patients (n=103)
Gender
male / 71
female / 32
Age (years)
median / 73.3
range / 21.9-89.5
CMML
-1 / 65 (63.1%)
-2 / 38 (36.9%)
Karyotype
normal / 71 (68.9%)
aberrant / 27 (26.2%)
no data available / 5 (4.8%)
WBC count x 10^9/L
median / 15.6
range / 2.4-160
no data available / 8 (7.8%)
Platelet count x 10^9/L
median / 91
range / 1,119-14
no data available / 19 (18.4%)
Hemoglobin level (g/L)
median / 11.2
range / 4-17
no data available / 19 (18.4%)
T-ALL
Patients (n=99)
Gender
male / 72 (72.7%)
female / 27 (27.3%)
Age (years)
median / 41.2
range / 18.8-87.7
T-ALL subtype
early / 41/89 (46.1%)
pre / 36
pro / 5
cortical / 44/89 (49.4%)
mature / 4/89 (4.5%)
no data available / 10 (11.2%)
Karyotype
normal / 29/93 (31.2%)
aberrant / 64/93 (68.8%)
no data available / 6 (6.1%)
WBC count x 10^9/L
median / 31.8
range / 1.6-450.0
no data available / 21 (21.2%)
Platelet count x 10^9/L
median / 86
range / 1.5-946.0
no data available / 27 (27.3%)
Hemoglobin level (g/L)
median / 11.5
range / 5.31-16.6
no data available / 29 (29.3%)
Clinical follow-up data
in total / 451/511 (88.3%)
AML / 185
MDS / 104
CMML / 88
T-ALL / 74

Supplementary Table 2

The table shows the identified mutations within the MLL cohort. The column cohort indicates the four different entities (AML, CMML, MDS, and T-ALL) analyzed in this study. Mutation shows the variation effect on DNA level. Consequence shows the effect on protein level. Type displayed the mutation category (M: Missense; N: Nonsense; F: Frame-shift; I: In-frame; S: Splice-site). Allele status indicates whether the variation affects one allele (monoallelic) or both alleles (biallelic). The genotypestatus is shown in columnI.Protein domain displays the location of a sequence variation within a functional domain. %mutated gives the mutation load. Column Mutation predictions displays the result of the prediction result of MutationTaster.

cohorte / exon / mutation / consequence / type / Allele status:
1=monoallelic; 2=biallelic / Genotype status:
1=homo; 2=hetero / Protein
domain / result / % mutated / Mutation prediction
AML / 9 / c.1066delC / p.Gln356Argfs*51 / F / 1 / 2 / 1 / positiv / 13.5 / disease_causing
AML / 9 / c.1118T>G / p.Leu373Arg / M / 1 / 2 / 0 / positiv / 30.0 / disease_causing
AML / 12 / c.1430_1431delAG / p.Glu477Alafs*14 / F / 1 / 2 / 0 / positiv / 24.2 / disease_causing
AML / 13 / c.1502A>G / p.Asn501Ser / M / 1 / 2 / 0 / positiv / 21.0 / disease_causing
AML / 14 / c.1609_1618dupTGCACCAT / p.Cys540Leufs*9 / F / 2 / 2 / 2 / positiv / 35.0 / disease_causing
AML / 14 / c.1609T>A / p.Cys537Ser / M / 2 / 2 / 2 / positiv / 48.0 / disease_causing
AML / 14 / c.1627G>T / p.Gly543Cys / M / 1 / 2 / 2 / positiv / 48.0 / disease_causing
AML / 14 / c.1627G>T / p.Gly543Cys / M / 1 / 2 / 2 / positiv / 48.5 / disease_causing
AML / 14 / c.1627G>T / p.Gly543Cys / M / 1 / 2 / 2 / positiv / 47.0 / disease_causing
AML / 14 / c.1640T>A / p.Leu547His / M / 1 / 2 / 2 / positiv / 48.0 / disease_causing
AML / 14 / c.1646G>A / p.Cys549Tyr / M / 2 / 2 / 2 / positiv / 31.0 / disease_causing
AML / 16 / c.1904G>A / p.Arg635Gln / M / 1 / 2 / 3 / positiv / 29.0 / disease_causing
AML / 16 / c.1904G>A / p.Arg635Gln / M / 1 / 2 / 3 / positiv / 36.0 / disease_causing
AML / 17 / c.1958T>G / p.Leu653Trp / M / 1 / 2 / 3 / positiv / 50.8 / disease_causing
AML / 17 / c.1961delG / p.Gly654Alafs*51 / F / 2 / 2 / 3 / positiv / 31.0 / disease_causing
AML / 17 / c.2007dupC / p.Ile670Hisfs*43 / F / 2 / 3 / positiv / 33.0 / disease_causing
AML / 17 / c.2056G>C / p.Asp686Arg / M / 1 / 2 / 3 / positiv / 37.0 / disease_causing
AML / 18 / c.2102T>C / p.Phe701Ser / M / 2 / 3 / positiv / 62.0 / disease_causing
AML / 18 / c.2120G>A / p.Gly707Asp / M / 1 / 2 / 3 / positiv / 46.2 / disease_causing
AML / 18 / c.2120G>A / p.Gly706Glu / M / 2 / 2 / 3 / positiv / 45.7 / disease_causing
AML / 18 / c.2129G>A / p.Cys710Tyr / M / 1 / 2 / 3 / positiv / 47.4 / disease_causing
AML / 18 / c.2141C>G / p.Ser714Cys / M / 1 / 2 / 3 / positiv / 47.0 / disease_causing
AML / 18 / c.2146G>A / p.Val716Ile / M / 1 / 2 / 3 / positiv / 45.0 / disease_causing
AML / 19 / c.2195T>C / p.Phe732Ser / M / 1 / 2 / 3 / positiv / 43.0 / disease_causing
AML / 19 / c.2261T>G / p.Leu754Arg / M / 1 / 2 / 3 / positiv / 50.0 / disease_causing
AML / 19 / c.2299delA / p.Arg767Glyfs*12 / F / 1 / 2 / 3 / positiv / 30.0 / disease_causing
AML / 19 / c.2322+1G>T / p.splice-site mutation / S / 1 / 2 / 3 / positiv / 25.0 / disease_causing
AML / 20 / c.2339T>C / p.Ile780Thr / M / 1 / 2 / 3 / positiv / 43.0 / disease_causing
AML / 20 / c.2396C>G / p.Pro799Arg / M / 1 / 2 / 3 / positiv / 45.0 / disease_causing
AML / 20 / c.2396C>G / p.Pro799Arg / M / 1 / 2 / 3 / positiv / 41.0 / disease_causing
AML / 22 / c.2479_2483delTTCAG / p.Phe827Glnfs*26 / F / 1 / 2 / 3 / positiv / 48.3 / disease_causing
AML / 23 / c.2635A>G / p.Asn879Asp / M / 1 / 2 / 3 / positiv / 46.0 / disease_causing
AML / 23 / c.2640G>T / p.Met880Ile / M / 1 / 2 / 3 / positiv / 47.0 / disease_causing
AML / 23 / c.2641A>C / p.Ser881Arg / M / 2 / 3 / positiv / 44.3 / disease_causing
AML / 23 / c.2644C>T / p.Arg882Cys / M / 1 / 2 / 3 / positiv / 45.0 / disease_causing
AML / 23 / c.2644C>T / p.Arg882Cys / M / 1 / 2 / 3 / positiv / 21.0 / disease_causing
AML / 23 / c.2644C>T / p.Arg882Cys / M / 1 / 2 / 3 / positiv / 50.0 / disease_causing
AML / 23 / c.2644C>T / p.Arg882Cys / M / 1 / 2 / 3 / positiv / 47.0 / disease_causing
AML / 23 / c.2644C>T / p.Arg882Cys / M / 1 / 2 / 3 / positiv / 44.9 / disease_causing
AML / 23 / c.2644C>T / p.Arg882Cys / M / 1 / 2 / 3 / positiv / 34.5 / disease_causing
AML / 23 / c.2644C>T / p.Arg882Cys / M / 1 / 2 / 3 / positiv / 38.0 / disease_causing
AML / 23 / c.2644C>T / p.Arg882Cys / M / 1 / 2 / 3 / positiv / 43.0 / disease_causing
AML / 23 / c.2644C>T / p.Arg882Cys / M / 1 / 2 / 3 / positiv / 23.0 / disease_causing
AML / 23 / c.2644C>T / p.Arg882Cys / M / 1 / 2 / 3 / positiv / 31.1 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 47.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 47.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 42.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 48.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 44.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 39.8 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 46.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 41.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 42.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 45.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 46.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 42.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 43.6 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 47.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 49.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 39.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 47.5 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 48.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 40.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 48.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 45.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 41.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 47.8 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 41.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 46.0 / disease_causing
AML / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 43.7 / disease_causing
AML / 23 / c.2645G>C / p.Arg882Pro / M / 1 / 2 / 3 / positiv / 42.4 / disease_causing
AML / 23 / c.2696G>A / p.Arg899His / M / 2 / 3 / positiv / 43.0 / disease_causing
AML / 8 / c.915G>A / p.Trp305* / N / 1 / 2 / 1 / positiv / 44.0 / disease_causing
AML / 8 / c.923G>A / p.Gly308Asp / M / 1 / 2 / 1 / positiv / 50.0 / disease_causing
AML / 8 / c.976C>A / p.Arg326Ser / M / 2 / 1 / positiv / 35.0 / disease_causing
CMML / 13 / c.1502A>G / p.Asn501Ser / M / 2 / 0 / positiv / 52.0 / disease_causing
CMML / 15 / c.1675delT / p.Cys559Alafs*92 / F / 2 / 2 / positiv / 40.0 / disease_causing
CMML / 19 / c.2196dupT / p.Glu733* / N / 2 / 3 / positiv / 39.0 / disease_causing
CMML / 19 / c.2206C>G / p.Arg736Gly / M / 2 / 3 / positiv / 36.0 / disease_causing
CMML / 23 / c.2644C>T / p.Arg882Cys / M / 2 / 3 / positiv / 39.0 / disease_causing
CMML / 23 / c.2645G>A / p.Arg882His / M / 2 / 3 / positiv / 46.0 / disease_causing
CMML / 23 / c.2723A>G / p.Tyr908Cys / M / 2 / 0 / positiv / 42.0 / disease_causing
MDS / 13 / c.1502A>G / p.Asn501Ser / M / 2 / 0 / positiv / 51.0 / disease_causing
MDS / 14 / c.1627G>T / p.Gly543Cys / M / 2 / 2 / positiv / 50.0 / disease_causing
MDS / 14 / c.1628G>A / p.Gly543Asp / M / 2 / 2 / positiv / 44.0 / disease_causing
MDS / 14 / c.1646delinsAA / p.Cys549* / N / 2 / 2 / positiv / 40.0 / disease_causing
MDS / 16 / c.1901T>A / p.Ile634Asn / M / 2 / 3 / positiv / 14.0 / disease_causing
MDS / 17 / c.2049C>A / p.Tyr683* / N / 2 / 3 / positiv / 42.0 / disease_causing
MDS / 18 / c.2086delC / p.Gln696Argfs*9 / F / 2 / 3 / positiv / 39.0 / disease_causing
MDS / 18 / c.2099C>A / p.Pro700Gln / M / 2 / 3 / positiv / 21.0 / disease_causing
MDS / 19 / c.2192T>A / p.Phe731Tyr / M / 2 / 3 / positiv / 44.0 / disease_causing
MDS / 19 / c.2255_2257delTCT / p.Phe752_Trp753delinsTrp / I / 2 / 3 / positiv / 47.0 / disease_causing
MDS / 19 / c.2311C>T / p.Arg771* / N / 2 / 3 / positiv / 11.0 / disease_causing
MDS / 20 / c.2387G>A / p.Gly796Asp / M / 2 / 3 / positiv / 45.0 / disease_causing
MDS / 20 / c.2392_2393delCT / p.Leu798Serfs*13 / F / 2 / 3 / positiv / 37.0 / disease_causing
MDS / 21 / c.2478G>C / p.Lys826Asn / M / 2 / 3 / positiv / 8.0 / disease_causing
MDS / 23 / c.2683G>A / p.Val895Met / M / 1 / 3 / positiv / 87.0 / disease_causing
T-ALL / 9 / c.1115T>A / p.Val372Asp / M / 2 / 2 / 0 / positiv / 55.0 / disease_causing
T-ALL / 14 / c.1555-1G>C / p.splice-site mutation / S / 2 / 2 / 0 / positiv / 50.0 / disease_causing
T-ALL / 14 / c.1578C>G / p.Tyr526* / N / 2 / 1 / 0 / positiv / 98.0 / disease_causing
T-ALL / 14 / c.1627G>T / p.Gly543Cys / M / 1 / 2 / 2 / positiv / 47.0 / disease_causing
T-ALL / 14 / c.1640T>A / p.Leu547His / M / 1 / 2 / 2 / positiv / 44.0 / disease_causing
T-ALL / 15 / c.1717_1732delinsT / p.Gln573* / N / 1 / 2 / 2 / positiv / 34.0 / not predictible (too large deletion)
T-ALL / 15 / c.1843C>T / p.Gln615* / N / 2 / 2 / 0 / positiv / 37.0 / disease_causing
T-ALL / 16 / c.1930G>A / p.Ala644Thr / M / 2 / 3 / positiv / 60.0 / disease_causing
T-ALL / 16 / c.1936G>C / p.Gly646Arg / M / 2 / 2 / 3 / positiv / 37.0 / disease_causing
T-ALL / 18 / c.2089G>T / p.Glu697* / N / 2 / 1 / 3 / positiv / 95.0 / disease_causing
T-ALL / 18 / c.2158C>T / p.Arg720Cys / M / 1 / 2 / 3 / positiv / 34.0 / disease_causing
T-ALL / 19 / c.2183delG / p.Gly728Alafs*51 / F / 2 / 3 / positiv / 33.0 / disease_causing
T-ALL / 19 / c.2207G>A / p.Arg736His / M / 2 / 2 / 3 / positiv / 18.0 / disease_causing
T-ALL / 19 / c.2226dupG / p.Pro743Alafs*6 / F / 2 / 2 / 3 / positiv / 46.0 / disease_causing
T-ALL / 20 / c.2375G>A / p.Arg792His / M / 2 / 3 / positiv / 60.0 / disease_causing
T-ALL / 22 / c.2501C>G / p.Thr834Ser / M / 2 / 3 / positiv / 20.0 / disease_causing
T-ALL / 22 / c.2567_2568delAG / p.Glu856Glyfs*7 / F / 2 / 1 / 3 / positiv / 65.0 / disease_causing
T-ALL / 23 / c.2644C>T / p.Arg882Cys / M / 1 / 2 / 3 / positiv / 56.0 / disease_causing
T-ALL / 23 / c.2644C>T / p.Arg882Cys / M / 1 / 2 / 3 / positiv / 48.0 / disease_causing
T-ALL / 23 / c.2645G>A / p.Arg882His / M / 2 / 2 / 3 / positiv / 47.0 / disease_causing
T-ALL / 23 / c.2645G>A / p.Arg882His / M / 1 / 2 / 3 / positiv / 16.0 / disease_causing
T-ALL / 23 / c.2711C>T / p.Pro904Leu / M / 2 / 3 / positiv / 43.0 / disease_causing
T-ALL / 8 / c.941G>A / p.Trp314* / N / 2 / 1 / positiv / 53.0 / disease_causing
T-ALL / 8 / c.976C>A / p.Arg326Ser / M / 2 / 1 / positiv / 42.0 / disease_causing

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